Hypoxic stress induces changes at the molecular level that may be important for aspects of tumor progression and therapy. Specifically, the increased synthesis of a set of proteins termed oxygen-regulated proteins (ORPs) is induced by severe hypoxia. This induction and subsequent decay upon; reoxygenation are temporally correlated with the occurrence of drug resistance and radiosensitivity. The original group of five documented ORPs has now been expanded to include at least four other polypeptides. We have identified three ORPs, including heme oxygenase- 1 as an ORP 33 and ORPs 80 and 100 as glucose-regulated proteins 78 and 94, respectively. We also have data suggesting that hypoxia causes increased synthesis of the epidermal growth factor receptor. The ORPs are a unique group of stress proteins that are probably induced in response to disruption of cellular energy metabolism. The putative hypoxia-responsive elements of the ORP genes have not been investigated. Experiments are proposed here to study the mechanisms of induction and regulation of these hypoxia-induced stress proteins by using heme oxygenase-1 as a model. We believe that these studies will contribute to a better understanding of the genetic elements involved in the early events of cellular adaptation to hypoxia and may aid in the design of new strategies for therapeutic intervention. We also propose to isolate other prominent ORPs and to determine the identities of three of them by peptide sequencing or screening of cDNA libraries. Selection of specific ORPs for detailed studies will be determined by criteria outlined in Experimental Design and Methods. Purified proteins will also be used to prepare antibodies for visualizing the intracellular locations of the ORPs. We anticipate that these studies of ORP characterization and regulation will lead to a fundamental understanding of the role of hypoxic microenvironments in tumor biology.